Generation of reactive oxygen species (ROS) under physiological conditions is required for normal force production in skeletal muscle. However, high levels of ROS are observed during intense physical exercise and promote contractile dysfunction, resulting in muscle weakness and fatigue. There is growing evidence that sub-cellular site-specific ROS production governs the beneficial vs. damaging effects of ROS. NADPH oxidase (Nox2) is an enzyme complex that generates ROS. Ongoing work by PI has found that increased ROS generation from Nox2 during intense contractile activity contributes to fatigue. We have strong evidence that Src tyrosine kinase acts as a redox switch to activate Nox2. The central hypothesis of this proposal is that Nox2 and Src act through a feed-forward pathway leading to excessive ROS production and contractile dysfunction. We will use genetically modified mice lacking Nox2, genetically encoded site-specific redox sensors and a novel tool to measure force and Ca2+ transients simultaneously in living skeletal muscle fibers to address the following specific aims: 1. Define the mechanisms by which contractile activity increases Nox2 dependent ROS production in skeletal muscle, 2. Determine whether Src serves as a redox switch to modulate activity dependent ROS production, and 3. Assess the role of Nox2 and Src on cytosolic [Ca2+ ]i transients and force generation. The proposed research will identify the sub-cellular sites of ROS production and elucidate the signaling pathways that control ROS regulation of microdomain Ca2+ signaling in skeletal muscle. Furthermore, it will lay the foundation for the development of targeted antioxidant therapy to combat the deleterious effects of overproduction of ROS seen in muscle fatigue and disease.